Bacterial contamination and biofilm formation on abiotic surfaces and strategies to overcome their

21 Pages·2017·1.03 MB·English

Journal of Materials and JMES, 2017 Volume 8, Issue 9, Page 3326-3346
Environmental Sciences
ISSN: 2028-2508
CODEN: JMESCN
http://www.jmaterenvironsci.com /

Copyright © 2017,
University of Mohammed Premier
Oujda Morocco

Bacterial contamination and biofilm formation on abiotic surfaces and
strategies to overcome their persistence

S.O. Khelissa 1, 2, M. Abdallah 2, C. Jama 1, C. Faille 2, N-E Chihib 2,*
1 Université deLille1, CNRS, ENSCL, UMR 8207-UMET-PSI, Avenue Dimitri Mendeleïev, CS 90108 59652 Villeneuve
d'Ascq, France
2 Université de Lille1, CNRS, INRA, UMR 8207-UMET-PIHM, 369 rue Jules Guesde, CS 20039, 59651 Villeneuve d'Ascq,
France

Abstract
Received, 26 Apr 2017
Revised, 15 May 2017 Abiotic surfaces are vulnerable to bacterial adhesion and to biofilm formation.

Accepted 17 May 2017 Therefore, it is necessary to understand the parameters that influence bacterial adhesion
to find out solutions against cell adhesion and biofilm formation. The ability of

Keywords pathogenic bacteria to adhere and to form biofilms on abiotic surfaces represents a
 Biofilm resistance, major health safety problem. Bacteria embedded in biofilms are more resistant to

sanitizing agents than those growing under planktonic state. In fact, surface
 adherent cells,
contamination by these pathogens is enhanced by favorable environmental conditions

 abiotic surface,
encountered in food and health sectors. Thus, the understanding of bacterial adhesion
 anti-biofilm and biofilm formation on abiotic surfaces is of interest to setup efficient anti-biofilm

strategies strategies. In this context, this review highlights the main factors controlling the
bacterial adhesion and biofilm formation on abiotic surfaces. It also describes the

current and emergent strategies used to eradicate and prevent the biofilm formation on
[email protected]
Tel: [phone] the most frequently used abiotic surface.

1. Introduction
Microbial adhesion onto abiotic surfaces and therefore the biofilm formation are considered serious issues,
regarding their economical and public health consequences in many sectors, such as food-processing and health-
care ones. The presence of pathogenic microorganisms on food sector facilities represents a severe potential
health risk to consumers. Contaminated food contact surfaces promote contamination of food products which
leads to Food-Borne Diseases (FBDs) [1]. In 2014, 864 FBD outbreaks were reported in the United States (US)
resulting in 13,246 illnesses, 712 hospitalizations, 21 deaths, and 21 food recalls [2].
According to the European Food Safety Authority (EFSA) and the European Centre for Disease Prevention and
Control (ECDC), a total of 5,251 food-borne outbreaks, including water-borne outbreaks, were reported in the
European Union (EU) in 2014. Overall, 45,665 human cases, 6,438 hospitalizations and 27 deaths were
reported. The evidence supporting the link between human cases and food vehicles was strong in 592 outbreaks
[3]. In healthcare sector, orthopedic implant surface bacterial contamination is responsible for nosocomial
infections also called Healthcare-Associated Infections (HAIs). Such infections are defined as infections that
occurred during a hospitalization and are not present prior to hospital admission. Generally, nosocomial
infections appear after prosthetic and implant surgery by handling contaminated or non-sterile devices. In
France, from 1999 to 2006, 14,845 surgical site infections were reported involving 964,128 patients in 838
participating hospitals [4]. HAIs and FBDs are responsible for high critical economic losses. In fact, the direct
cost of the HAIs was up to $16.6 billion in the US hospitals [5]. It has been reported that the resulting
aggregated annual cost of FBD was $77.7 billion [6]. Generally, microorganisms live attached to surfaces and
form biofilm [7]. When bacteria grow within a biofilm they gain several advantages, including enhanced
resistance to antimicrobial agents [8]. Biofilms represent a threat to public health when found in food [9] and
medical sector [10]. In addition, biofilms are also of concern in different other sectors such as maritime
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environment [11], water systems [12] and in oil pipes industries [13]. Their formation results in heavy costs in
cleaning and maintenance. The persistence of biofilm in both food and medical sectors may constitute a
reservoir for pathogens which increase the occurrence of HAIs and FBDs. Therefore, it is necessary to
investigate different strategies in order to reduce the bacterial adhesion and the formation of biofilm.
Disinfection is an important used strategy to control biofilm formation and to avoid infection transmission.
Other strategy requires designing abiotic surfaces able to hamper the bacterial adhesion and therefore the
biofilm formation.
Rather than developing new materials, another promising way is surface modification of existing surfaces [14]
by grafting functional chemical groups or antibacterial molecules inhibiting bacterial adhesion [15]. However,
the main challenge here is the durability of the treated surface [16]. Thus, setting up antimicrobial surfaces
could be very useful for food processing equipment to enhance the food safety and in biomedical sector to
prevent microbial colonization on hospital surfaces. To achieve such challenge, the choice of appropriate
antimicrobial molecules and surface modification techniques is required. In addition, a deep understanding of
the interaction between three main components: the bacterial cell, the attachment surface, and the environmental
parameters is needed. In this regard, the goal of this review is to discuss the impact of bacterial adhesion and
biofilm formation on abiotic surfaces. In addition, we attempt to highlight the strategies and approaches
commonly applied in order to prevent bacterial adhesion and by the way biofilm formation.
2. Main pathogenic bacteria associated with FBDs and HAIs
2.1. Food-borne diseases
Bacteria are all around us, in the air, on surfaces and in/on the human body. Bacteria are often harmless but
some of them can be pathogenic for humans. In natural, industrial, hospital and domestic environments, there
are many persistent pathogenic bacteria such as Listeria monocytogenes, Staphylococcus aureus, Salmonella
spp., Escherichia coli, Pseudomonas aeruginosa and Bacillus cereus which have serious economical and public
health consequences. The contamination of abiotic surfaces with these pathogens leads to human infections
worldwide [17]. L. monocytogenes has been involved as causative agent of FBDs due to its ubiquitous nature
and its ability to grow under hostile conditions [18,19]. This bacterium is frequently associated with FBDs
outbreaks that are characterized by wide spread distribution and relatively high mortality rates. Listeriosis, a
serious infection, is usually caused by eating contaminated food. The disease primarily affects older adults,
pregnant women, newborns, and adults with weakened immune systems. According to the EFSA and the
ECDC, the number of confirmed human listeriosis cases in the EU increased slightly to 1,642 in 2012 compared
with 2011 [20]. This number includes 198 death cases, which represents the highest number of fatal cases
reported since 2006. According to this study, France is the most affected country with up to 63 fatal reported
cases. Thus, the EU fatality case rate was 17.8 % among the 1,112 confirmed cases (67.7 % of all confirmed
cases) [20]. The CDC estimates that about 1,600 illnesses and 260 death cases due to listeriosis occur annually
in the US [21]. The worst listeriosis outbreak in the US history has occurred in 2011 and it was associated with
consumption of cantaloupe from a single farm. In fact, 147 illnesses, 33 deaths, and 1 miscarriage were reported
in 28 states [22]. This psychotropic microorganism is able to grow at refrigeration temperatures as low as 2 to
4°C [23] and to contaminate the food-processing environment. Contamination of food with L. monocytogenes
seems to occur most frequently during the food-processing due to the ability of this bacterium to attach to
Stainless Steel (SS) and other abiotic surfaces [24] and form biofilm [25]. In addition, L. monocytogenes has
been isolated from various surfaces in dairy and meat processing environments [26]. FBDs are also commonly
caused by Gram-positive enterotoxigenic S. aureus [27]. S. aureus is an ubiquitous bacterium which can be
found in the air, dust, sewage, humans and animals. In France, food poisoning cases associated with S. aureus
have been listed in 2012 as the first cause of food-borne outbreaks [28]. In fact, 300 of 1,288 reported food-
borne outbreaks (23%) were due to this pathogen [28]. S. aureus is able to adhere and form biofilm in food
processing plants [29]. Despite the inactivation of S. aureus by heating the food prior to consumption, this
bacterium can still induce intoxication. In fact, staphylococcal enterotoxins remain stable since they resist to
extreme environmental conditions (freezing, drying, heat treatment, low pH and proteolytic enzymes)
[27,30,31]. According to the EFSA and ECDC [32], in 2011, 6.1 % of all food-borne outbreaks in the EU were
caused by staphylococcal toxins. This represent an increase of 25.9 % compared to 2010 (274 outbreaks) and
was mainly due to the fact that France has reported 290 outbreaks in 2011 compared with 220 in 2010. In
France, S. aureus represents the second cause of FBDs after Salmonella with 1,361 cases [33]. Salmonella spp.
is the major food-borne pathogen for humans and animals worldwide. It has been reported that about 1.4 million
human salmonellosis cases occur in the US leading to more than 16,000 hospitalizations with nearly 600 deaths
and resulting in a high cost amounting to several billion dollars annually [34]. In 2012, the number of
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salmonellosis cases in humans decreased by 4.7% compared with 2011. A statistically significant decreasing
trend in the EU was observed over the period 2008-2012. A total of 91,034 confirmed human
salmonellosiscases were reported in 2012 [20]. The two most common Salmonella serovars, involved in food
poisoning outbreaks, are Typhimurium and Enteritidis [35]. Salmonella Enteritidis was the predominant serovar
associated with the Salmonella outbreaks accounting for 66 % of human cases involved in these outbreaks
followed by Salmonella Typhimurium which has been associated with 16.9 % of cases. The persistence of
Salmonella in food processing environment, despite the cleaning procedures, could lead to microbial cross-
contamination and to biofilm formation [36,37]. In fact, several studies have demonstrated the ability of
Salmonella to form biofilms on abiotic surfaces such as SS [38], plastic [39] and rubber [40]. Generally, once
attached, these pathogens may produce resistant biofilms constituting a reservoir for cells which, once detached,
contaminate food products continuously. In addition, it is now established that in natural and man-made
ecosystems, more than 99.9% of micro-organisms live attached to surfaces and form a specific and complex
structure called biofilm. E. coli strains are common bacteria of the gastrointestinal tract [41]. Some E. coli
strains are able to produce toxins that induce serious human infections [41]. Grass-fed cattle are the main
reservoir of such E. coli strains. Their faeces might contaminate the meat during slaughter and thus act like
microbial carrier which might end up contaminating other foods (e.g. milk, vegetables) and water. Outbreaks
due to E. coli 0157:H7 have been associated primarily with consumption of undercooked beef meat, but also
other foods have been involved as contamination carrier [42]. In fact, cross-contamination of foods can occur in
food-processing plants and during subsequent handling and preparation, resulting in a wide range of foods being
involved in E. coli O157:H7 outbreaks [43,44]. In 2011, the ECDC have reported 2,495 food-borne outbreaks
caused by the pathogenic E. coli including 54 deaths in the EU [32].

2.2. Nosocomial infections
According to the ECDC the most frequently reported HAI type was pneumonia and other lower respiratory tract
infections, representing 25.7% of all reported HAIs [45]. The second most frequently reported type of HAI was
surgical site infection (18.9%) followed by urinary tract infection (17.2%), bloodstream infection (14.2%) and
gastro-intestinal infection (7.8%) [45]. S. aureus and P. aeruginosa are in the top four microorganisms most
frequently isolated from these HAIs in the EU [45]. P. aeruginosa is found in various environmental niches
including soil, water, plants, and hospital environments [46]. Despite the advances in health care and the
improvement of strict disinfection procedures, P. aeruginosa is among the most dreaded Gram-negative
pathogens in hospital setting and is the one of main causes of nosocomial infections [47]. According to the
National Healthcare Safety Network (NHSN), P. aeruginosa was involved in 8% of HAIs in the US hospitals
[48]. Moreover, in 2013, the CDC reported that about 51,000 health-care-associated P. aeruginosa infections
occur in the US each year. More than 6,000 (13%) of these are multi-drug-resistant with roughly 400 deaths per
year [49]. In the EU, P. aeruginosa represents 8.9% of total pathogens associated with nosocomial infections
[45]. P. aeruginosa is an important cause of infection among patients with impaired immune systems. In 2012,
high percentages of Multi-Drug Resistant (MDR) P. aeruginosa isolates were reported in several countries,
especially in Southern and Eastern Europe. Combined resistance was common, with 14% of the isolates
reported as resistant to at least three different antimicrobials [50]. Another bacterium causing similar problems
is the Gram-positive S. aureus. Besides to being responsible for food poisoning outbreaks, this species has been
recognized as an important pathogen which causes different serious human diseases [51]. S. aureus in its
methicillin-resistant form (MRSA) is a major cause of anti-microbial resistant health-care associated infections
worldwide. MRSA remains a public health priority in the EU, as the percentage of MRSA is still above 25% in
seven of 29 reporting countries [52]. In the EU the number of patients acquiring health-care-associated
infections in acute care hospitals has been estimated at 4.1 million each year [53]. S. aureus is the most involved
pathogen in bloodstream infections in the US. According to the NHSN, this bacterium is associated with 15% of
total HAIs reported between 2011 and 2012 in the US [54].

3. How bacteria adhere to surfaces and form biofilms?
Biofilm formation is a complex process which gives bacteria a better resistance to cleaning agents than bacteria
growing under planktonic form [7]. Biofilm is a community of microorganisms in which cells stick to a surface
and to each other (Figure 1).
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Figure 1: Scanning electron microscopy image of biofilm produced by Staphylococcus aureus CIP 4.83 on 316L
stainless steel after 24 h incubation at 37°C. The scale bars in the images are 2.5 μm (a) and 10 µm (b).
This cell cluster is marked by the secretion of extracellular matrix (Figure 2) with adhesive and protective
properties [1,7].

Figure 2: Scanning electron microscopy image of Escherichia coli
biofilm on Teflon. The arrows in the image point to the extracellular
matrix enclosing the Escherichia coli biofilm bacteria (colored in
pink). The scale bar in the image is 10 μm.

Biofilm formation requires different steps and there are a number of mechanisms by which many microbial
species may come closely in contact with a surface, attach and promote cell-cell interactions in order to grow
and form biofilms. Theses mechanisms have been widely described [55]. The different steps leading to biofilm
formation are now well understood. The adsorption of bacteria or reversible adhesion to the surface is the first
step of biofilm formation. It is triggered when the microorganisms approach the surface over 50 nm, through
van der Waals interactions. Then, when the distance is between 10 and 20 nm, more non-covalent forces such as
hydrophobic, acid-base and electrostatic interactions get involved in the adhesion process. As the distance
decreases the adhesion becomes irreversible, and at less than 0.5 nm other specific interactions, also called
short-range interactions, are needed to attach bacteria to abiotic surfaces (Figure 3). In fact, bacteria have some
structural adhesins which are a part of the cellular envelope such as pili, Fimbria and flagella that enhance
cellular adhesion. These structures create bridges between cells and surfaces and allow overcoming unfavorable
conditions in order to strongly anchor bacteria to abiotic surfaces [56].
Once the irreversible adhesion is established, bacteria start synthesizing insoluble exopolysaccharides (EPS).
Within hours of EPS accumulation, bacteria get entrapped in a complex protecting extracellular matrix and form
a mature biofilm that provides protective environments against antibacterial agents and antibiotics [57]. Indeed,
this EPS matrix makes traditional surface cleaning procedures and application of detergents or biocides on
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materials in contact with food not fully efficient to eliminate mature biofilms [58]. Therefore, one of the most
effective strategies to limit biofilm formation is to prevent or restrict bacterial adhesion on surfaces. Bacterial
adhesion on abiotic surfaces and subsequent biofilm formation constitute a serious issue in several sectors such
as food industries, water canalizations and medical facilities. Indeed, bacteria find favorable conditions to
colonize surfaces and establish biofilms [7]. The persistence of biofilm in food, medical and other sectors
constitutes reservoirs for pathogens which increase the occurrence of HAIs and FBDs. Thus, it is necessary to
investigate different strategies in order to reduce the bacterial adhesion and the formation of biofilm.

Figure 3: The reversible bacterial adhesion consists in the initial attraction of the bacterial cells to the surface through
the effects of non-specific physical forces (distance > 50 nm between bacterial cells and surfaces). The irreversible
adhesion is achieved through the effects of the specific (short-range) interactions (distances < 5 nm, with involvement
of hydrogen bonding, ionic and dipole interactions, hydrophobic interactions and bacterial structural adhesins).

4. Food-borne infections and adherent cells
Food contact surfaces and equipments are considered a serious factor contributing to contamination of foods if
not properly cleaned [59]. In addition, surface contamination may lead to biofilm formation which enhances the
capacity of food-borne bacteria to survive stress conditions encountered within food processing environments
[60]. Surface contamination by pathogenic bacteria results in serious food-borne outbreaks generating a
considerable disease burden and also economic losses [61]. The economic cost of food-borne outbreaks is
highly affecting the US economy at a cost of 50 to 80 billion US dollar annually [62]. Other statistics has
estimated that the total burden of FBDs was 152 billion US dollar [63]. In Australia and New Zealand, the cost
of food-borne outbreaks has been estimated at 1,289 billion and 86 million US dollar respectively per year
[64,65]. In Sweden, the annual cost of food-borne outbreaks was estimated to be 171 million US dollar [66]. In
this regard, globalizing of food market with worldwide transportation makes food safety a major priority in
order to prevent spreading of pathogenic bacteria and the emergence of food poisoning outbreaks worldwide. In
England and Wales, FBDs cause more than 2 million cases, 21,138 hospitalizations and 718 deaths per year
[67]. Pathogenic bacteria are able to adhere and form biofilms on various food contact surfaces [68,69]. It is
now established that the persistence of pathogenic bacteria on food contact surfaces, equipment and processing
environments, is a contributing factor in food-borne outbreaks, especially those involving L. monocytogenes, B.
cereus, S. aureus, E. coli and Salmonella spp. [70]. Equipment, utensils and cutting boards are likely to be the
key cross contamination routes as they become contaminated with pathogens from the handlers, sewage, water
and condensation caused by the faulty ventilation [71–73]. Therefore, it has been reported that in the United
Kingdom, 14 % of all food-borne illnesses involving S. aureus, E. coli, Salmonella enterica and L.
monocytogenes, may be due to inadequately cleaned cutting boards and knives [74]. According to the French
national health monitoring institute (InVS), 1,380 FBD outbreaks were reported in 2014, affecting 12,109
people, including 649 hospitalizations and 2 deaths. The three most frequently suspected pathogens were S.
aureus (30%), B. cereus (22%) and Salmonella spp. (15%). The French available data showed also that food
contact surfaces and equipment were up to 60 % involved in FBD outbreaks (2011) in collective and home
catering [75]. In fact, food industries represent a favorable environment for bacterial adhesion and biofilm
formation [76]. In the dairy, meat and sea-food industries food contact surfaces are often contaminated by
pathogenic bacteria including L. monocytogenes, S. aureus, Salmonella spp., B. cereus and E. coli [77–80].
Moreover, it has been reported that even after cleaning, E. coli bacterial densities up to 105 CFU/cm2 could be
recovered on food processing surfaces [81]. It has been mentioned that in small-scale facility producing
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traditional dry sausage, sixteen L. monocytogenes strains and nine Salmonella spp. subspecies were isolated
from the stuffing machines [82]. Moreover, many pathogenic bacteria such as B. cereus and S. aureus are often
isolated from the dairy, meat and sea-food industries surfaces [79]. In addition, a highest prevalence of sea gene
encoding for Staphylococcal Enterotoxin A (SEA) have been reported. The sea gene is the most common in
Staphylococcus-related food poisoning [83].

5. Nosocomial infections and adherent cells
Nosocomial infections contracted during hospitalization can lead to high morbidity and even mortality of
immune-depressed patients. Bacterial adhesion to medical devices surfaces and surgical sites is considered the
base of the pathogenic mechanism [84]. Bacterial risk is of major concern in the medical sector because of the
high rate of contamination of materials which are inserted into or in contact with the human body. Medical
implants such as urinary catheters, central venous catheters and implanted prosthetic devices are prone to
biofilm formation and represent a serious nosocomial infection source [85–87]. The issue starts when an
indwelling medical device is contaminated with pathogenic bacteria which may develop a biofilm. Once these
microorganisms irreversibly attach to devices introduced into a body, they start producing extracellular
polysaccharides to develop an infectious biofilm. Such infections are known nowadays as chronic polymer-
associated infection [88]. According to the National Nosocomial Infection Surveillance system of the CDC,
Blood-Stream Infections (BSIs) represent 90% of all nosocomial blood infections and they are always
considered to be device related if they happen after the insertion of an intravascular catheter [89]. Moreover,
intravascular catheters are one of the most common causes of nosocomial bacteremia. In fact, catheter-related
BSIs are affecting over 250,000 patients per year in US [90]. In this context, it has been shown by scanning and
transmission electron microscopy that almost all indwelling catheters are colonized by microorganisms
embedded in a biofilm matrix [91]. These biofilms may be located either on the lumen or on the outer surface of
the catheter [92]. The colonizing microorganisms may originate either from patient's skin micro-flora or other
micro-flora from health-care staff and contaminated facilities. Furthermore, staphylococci are recognized as the
most frequent causes of biofilm-associated infections [93]. The percentage of implant failure, due to infection
by three different groups of staphylococci: MRSA, Methicillin-Sensitive S. aureus (MSSA), and Coagulase-
Negative Staphylococci (CoNS), is of ca 2% of all implants, representing an average of 4500 incidents per year
[94]. Moreover, the prevalence of Ventilator-Associated Pneumonia (VAP) is 8 to 28% among patients who
received prolonged mechanical ventilation [95]. This results from the respiratory system colonization by the
endogenous flora or by exogenous pathogens acquired from the intensive care environment [96,97]. P.
aeruginosa is also considered one of the most frequently associated pathogen with HAIs. It has been identified
that healthcare water systems are associated with patient infections with P. aeruginosa in intensive care units
[98]. In fact, P. aeruginosa biofilms are likely to represent a potential reservoir source of nosocomial infection
when it colonizes water systems in healthcare facilities [98]. Besides to their fatality towards human, HAIs
represents a high economical cost. The annual direct medical cost of HAI to the US hospitals ranges from $28.4
to $33.8 billion [99]. In France the total cost of nosocomial infections in acute care units was estimated to be up
to €3.2 million per year [100].

6. Parameters controlling biofilm formation
Abiotic surfaces are vulnerable to biofilm formation. Therefore, it seems to be necessary to understand the
parameters that influence bacterial adhesion in order to find solutions against biofilm formation. Bacterial
adhesion to surfaces is likely to be related to three main parameters which are the physiochemical characteristics
of the bacterial cell and abiotic surfaces and finally the environmental conditions.

6.1. Role of the physiochemical characteristics of the bacterial cell surface in biofilm formation
The attachment of bacterial cells to abiotic surfaces is a process tightly related to several physiochemical forces
such as van der Waals, electrostatic, steric forces and hydrophilic ⁄ hydrophobic. Moreover, the physicochemical
surface properties of bacterial cells are determined by structures and molecules that are exposed on the cell
surface which control the attachment and biofilm formation. Here, the major bacterial cell structures will be
highlighted.

6.1.1. Role of bacterial cell surface structures
6.1.1.1. Flagella
Flagella have been generally considered major virulence factors mainly because of their motility property.
However, flagella are getting recognized to play other roles with more functions besides motility and
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chemotaxis. Recent studies have defined flagella as an effective bacterial surface compound in many additional
processes including adhesion, biofilm formation and virulence factor secretion [101]. Motility is considered a
virulence factor facilitating the colonization of abiotic surfaces by pathogenic bacteria. According to different
studies the flagellar motility is important for initial cell-to-surface contact leading to biofilm formation and
development [102,103]. Flagella can facilitate the attachment of bacteria to surfaces by overcoming the
repulsive forces that might hamper cell- to-surface contact. Thus, flagella are not only required for motility but
also plays an important role in surface sensing and the earliest steps of surface adhesion that leads to the
formation of a biofilm. E. coli and L. monocytogenes use flagella, pili, and membrane proteins to initiate
attachment [104]. The loss of these cell appendages changes their surface properties which may lead to
decreased attachment ability on some abiotic surfaces [105].
6.1.1.2. Fimbriae or pili
Fimbriae (or pili) are a group of rigid, straight, and filamentous proteinaceous structures composed of protein
subunits called pilin associated to the outer bacterial membrane surface [106]. Their role in biofilm formation on
abiotic surfaces is considered critical in the early stable cell-to-surface attachment. It has been showed that Type
1 and Type 3 fimbriae on Klebsiella pneumoniae strain surface are the main factors facilitating adherence and
the formation of a full-grown biofilm on abiotic surfaces [107,108]. Moreover, fimbriae have a critical role in P.
aeruginosa adhesion to SS, polystyrene and PolyVinyl Chloride (PVC) [109]. Type 1 fimbriae of E. coli
facilitate attachment on abiotic surfaces and promote biofilm formation. In fact, it has been reported that the
expression level of type 1 fimbriae had a direct effect on E. coli adhesion to surfaces [110]. Furthermore, it has
been reported that the presence of type I pili is essential for the initial attachment of E. coli to PVC [111]. In
fact, cells carrying lesions in genes encoding for the regulation or biogenesis of type I pili did not attach [111].
6.1.1.3. Extracellular polymeric substances (EPS)
The main composition of bacterial EPS includes polysaccharides, proteins, nucleic acids, lipids and
phospholipids [112]. The lipopolysaccharide (LPS) outer layer of Gram-negative bacteria affects the
bacterium’s susceptibility to disinfectants and influences the biofilm formation [113]. The pel genes encode
proteins with similarity to components involved in P. aeruginosa’s polysaccharide biogenesis. The pel gene
cluster is conserved in other Gram-negative bacteria and was previously identified in the P. aeruginosa PA14
strain as required for the production of a glucose-rich matrix material involved in the formation of a thick
pellicle and resistant biofilm. Indeed, mutation in pel genes may lead to an adherence defect [114]. For E. coli,
truncation of LPS affects the biosynthesis of Type 1 fimbriae and flagella resulting in a reduced adherence
[115]. Alterations in the peptidoglycan structure exposed at the surface of L. monocytogenes can also have an
effect on attachment [116]. Many bacteria produce EPSs which are an important constituent of the biofilm
extracellular matrix. Overproduction of EPS can even inhibit initial attachment of E. coli O157:H7 to SS [117].
Several studies targeting the cell-surface proteins have revealed the existence of a large group of cell-surface
protein called biofilm-associated proteins (Bap) on S. aureus. Recently, BapA was reported as necessary for
biofilm formation by Salmonella Enteritidis [118]. Moreover, in Salmonella biofilms, cellulose is the main
matrix EPS and represents the second component of EPS after the curli fimbriae. Cellulose is a β-1→4-D-
glucose polymer which is biosynthesized by the bcsABZC-bcsEFG genes (bacterial cellulose synthesis) [119],
two operons that are involved in cellulose biosynthesis in both Salmonella Enteritidis and Salmonella
Typhimurium respectively [120,121]. Colanic acid, another EPS belonging to capsular extracellular
polysaccharide, is essential for Salmonella Typhimurium biofilm. The importance of colanic acid in the biofilm
formation capacity of Salmonella strains unable to produce either curli fimbriae or cellulose have also been
confirmed [122].
6.1.2. Role of bacterial surface hydrophobicity in bacterial adhesion
In addition to the influence of the type of molecules expressed on the bacterial cell surface on the attachment to
solid surfaces, there is a correlation between bacterial surface hydrophobicity and adhesion. In general, bacteria
behave as hydrophobic particles. However, the degree of hydrophobicity depends on many parameters such as
the pH, the ionic strength of growth medium and the bacterial species [123]. It has been reported that S.
epidermidis strains with higher surface hydrophobicity adhered more than the ones with less surface
hydrophobicity to polyethylene [124]. Hydrophobicity of bacteria can be evaluated by contact angle
measurements, such as the sessile drop method or by their ability to adhere to hexadecane [125].
Khelissa et al., JMES, 2017, 8 (9), pp. 3326-3346 3332
6.1.3. Role of bacterial surface charge in bacterial adhesion
The surface charge of bacteria is another important physical factor for their adhesion [126]. Depending on their
surface groups’ ionization, bacteria acquire a surface electric charge in aqueous suspension. In fact, bacteria
have a net negative surface charge. The surface charge of bacteria varies according to bacterial species and is
influenced by the growth medium, the pH and the ionic strength of the suspending buffer, bacterial age and
bacterial surface structure [123]. The surface charge is usually characterized by the electrophoretic mobility
(zeta potential) [125]. However, the contribution of bacterial surface charge to bacterial adhesion has not been
clearly understood. The adhesiveness of S. epidermidis correlates directly with surface electro-negativity and
hydrophobicity while the adhesion of E. coli is inversely proportional to the degree of negative surface charge
but is not influenced by hydrophobicity [105].
6.1.4. Role of bacterial membrane potential in bacterial adhesion
Bacterial membrane potential is a physical characteristic that plays a dominant role in the adhesion of
microorganisms to abiotic surfaces. Surface potential mapping using Kelvin probe force microscopy showed
that the bacterial membrane potential is not the same on different material substrates [127]. The changes in
bacterial membrane potential have been considered a direct result of changes in cellular metabolism and motility
[127]. Adhesion has been shown to depend mainly on the pH, ionic strength of the suspending solution and of
material surface properties. Some studies had also established that the membrane potential plays an important
role in the bacterial adhesion on surfaces too [128].
6.2. Role of the physiochemical characteristics of the abiotic surface in biofilm formation
The main factors influencing bacteria adherence to abiotic surfaces include the physiochemical properties such
as surface energy and hydrophobicity, chemical composition of the solid surface and surface roughness [126].
6.2.1. Chemical composition of the solid surface
Bacterial adhesion to surface and biofilm formation depend on the solid surface chemistry. Surfaces can have
different functional groups that influence the bacterial attachment which depends also on the hydrophobicity
and charge of material [123]. S. aureus was found to adhere preferentially to metals and S. epidermis to
polymers [129]. This result may explain why S. epidermidis often causes polymer implant infection while S.
aureus is often the major pathogen in metal implant infections. The surface chemistry might be modified with
different types of coating. The most current is plasma coatings that considerably reduce bacterial adhesion to
surfaces [130]. Different studies have shown that the hydrophilicity of the native PVC was altered after
thiocyanation of PVC surface, resulting in the decrease of bacterial adhesion to this material [131]. It has been
reported that nisin-coated surfaces also inhibited the bacterial adhesion [132].
6.2.2. Surface topography and roughness
The relationship between bacterial adhesion and the surface topography was studied intermittently for 45 years
[133]. Thus different opinions on the effect of the surface roughness on bacterial adhesion and biofilm
formation have emerged. The food hygienic quality is closely related to the cleanability degree of equipment
used in the production lines. The roughness of SS is considered a primary factor in the attachment of bacteria
and biofilm formation [134]. The influence of material roughness on the bacterial adhesion has been
investigated closely. Many studies focusing on the topography of different types of surfaces have found that the
irregularities of abiotic surfaces enhance bacterial adhesion and biofilm formation whereas the smooth surfaces
decrease the ability of bacterial adhesion [135]. In fact, rough surfaces have a greater surface area and provide
for bacteria protective shelter against cleaning agents and more favorable sites for colonization (Figure 4) [76].
Moreover, porosity of materials has a significant effect on the bacterial attachment. It has been found that
implant site infection rates are different between porous and dense materials with porous materials having a
much higher rate. This shows that bacteria adhere and colonize the porous surface preferentially. Indeed,
bacteria adhere more to porous and grooved surfaces compared to dense and flat ones because of their larger
contact surface [135].
6.2.3. Surface energy and hydrophobicity degree
The physicochemical properties of abiotic surfaces in food processing industry are suspected to significantly
influence the biofilm formation mainly via the initial attachment of bacteria. In fact, the attachment of the
bacteria depends on the critical surface tension of the solid surface [136].
Khelissa et al., JMES, 2017, 8 (9), pp. 3326-3346 3333
Figure 4: Scanning electron microscopy image of Staphylococcus aureus CIP 4.83 adhesion on 316L
stainless steel. Stainless steel surface before (a) and after (b) the bacterial adhesion. Bacteria attach to the
crevices and align often along longitudinal scratches. The scale bars in the images are 25 μm (a) and 5 µm (b).

The surface energy of a solid surface is a direct indicator for interfacial attractive forces. The modification of the
surface energy of surfaces has a direct influence on the bacterial adhesion [137]. It has been reported that the
adhesion of S. xylosus depends on the physicochemical properties of the surface and ionic strength of the
surrounding medium [138]. It has been defined that hydrophobic interactions are the strongest of all non-
covalent interactions in biological systems [139]. Physicochemical forces involved in adhesion are dependent of
each other. The relationships between surface hydrophobicity and charge have been observed. A decrease in
surface charge is often accompanied by an increase in hydrophobicity [56]. Surface hydrophobicity has been
considered a determinant factor for microbial cell adhesion [140]. The concept of hydrophobicity opposes that
of surface wettability since hydrophobic surfaces present low wetting. Furthermore, hydrophilic surfaces
generally allow greater bacterial attachment and biofilm formation than hydrophobic ones [141]. Indeed, it has
been found that initial attachment of L. monocytogenes Scott A to SS was more rapid than to rubber [142].
Moreover, several studies have investigated the relation between the hydrophobicity degree and the bacterial
adhesion rate. The relationship between the hydrophobicity degree of different abiotic surfaces and the number
of attached S. epidermidis and Alcaligenes denitrificans cells have been assessed and results showed that the
adhesion rates increased with the surface hydrophobicity [143]. In the same context, Sheng et al. (2008) [144]
have reported that bacterial adhesion is lower on metal surfaces with reduced hydrophobicity.
6.3. Environmental conditions influencing bacterial adhesion
The physicochemical properties of both cell and material surfaces are very critical proprieties affecting the
adhesion of bacteria and the formation of biofilm [145]. Moreover, bacterial adhesion is an extremely
complicated process that is affected by many other factors including the environmental conditions (pH,
temperature, bacterial concentration, nutrient availability and the associated flow conditions) that need to be
controlled in order to find strategies against biofilm formation [68]. The number of attached bacteria is
significantly affected by the flow conditions and generally the number of attached bacteria decreases when
shears rates are high. Moreover, variations in pH value in the culture environment also influence bacterial
adhesion and the growth of biofilm [146]. The pH influences the cell surface hydrophobicity and better adhesion
to hydrophobic surfaces was found at pH in the range of the isoelectric point when bacteria are uncharged [147].
Therefore, pH influences bacterial adhesion by influencing the surface charge and changing surface
characteristics of the bacteria [55]. Moreover, variations in external pH can disturb the trans-membrane
electrochemical gradient and have a biocidal effect on the microorganisms. The growth temperature is also an
important condition for bacterial adhesion and biofilm formation as well as the presence of nutrient [1,148].
High growth temperature was found to increase the biomass and the attachment ability of bacteria probably, due
to the production of heat stress proteins associated with the cell surface [149,150]. Otherwise, different studies
concerning S. aureus biofilm formation have shown that temperature variation has no clear effect on the
biomass [151]. Thus, optimum temperature enhances the biofilm formation. Temperature also affects the
bacterial surface polymer composition which decreases at low temperature and reduces the adhesive properties
of bacteria [152]. Another important factor in biofilm formation is nutrient availability. In fact, nutrients
influence the surface charge of bacteria. For instance, glucose and lactic acid in the growth medium decreased
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the bacterial cell wall electro-negativity through the neutralization of the surface charge [153]. Thus, a
synergistic effect between the environmental factors may occur and affect biofilm formation.
7. Strategies to control biofilm formation and development
Virulence of microorganisms is often enhanced when imbedded into biofilm [154]. Unfortunately, in the
industrial fields, the availability of nutrient and water promotes the biofilm formation. In this regard, several
strategies have been proposed to control biofilm formation and to avoid biofouling. Ideally, preventing biofilm
formation would be a more logical option than treating it once established. Thus two major ways to control
biofilm formation can be adopted. The first one is based on the use of antimicrobial agents, physical forces,
enzymes, plant extracts, etc. to eradicate or disrupt already formed biofilms. The second strategy aims to
anticipate and prevent bacterial adhesion and therefore biofilm formation by modifying the physiochemical
properties of abiotic surfaces.
7.1. Eradication of biofilms
7.1.1. Cleaning and disinfecting of abiotic surfaces
In food processing industry, effective cleaning and disinfecting of equipment and surfaces is required to reduce
the bacterial contamination and produce safe products with acceptable shelf life and quality [155]. Cleaning is
the first step of sanitizing intended to reduce the number of pathogenic bacteria on surfaces before disinfecting
[155]. An efficient cleaning and disinfection procedure consists of a sequence of rinses using good quality water
with application of detergents and disinfectants [155]. Cleaning frequency must be clearly defined for each
process line (daily, after production runs, or more often, if necessary). Cleaning is an important step to minimize
microbial colonization of industrial food processing equipment. It seems to be of great importance to eliminate
as many micro-organisms as possible before applying a disinfectant [156]. In food and health sectors,
disinfectants are used for decontamination and to reduce the surface population of viable cells left after cleaning
in order to prevent microbial growth and biofilm formation on surfaces [112]. There are different kinds of
commercialized disinfectants such as alcohol based one, hypochloric solutions, aldehydes, hydrogen peroxide,
ozone and quaternary ammonium compounds (QACs) [1]. These disinfectants can be used in different sectors at
different concentrations [157]. The particularity of these antimicrobial agents is that they have more than one
target site. In fact, they can target the cytoplasmic constituents, the outer cell components and the cell
cytoplasmic membrane [158]. The activity and the efficiency of disinfectants against biofilms depend on several
chemical and physical factors such as concentration, pH, temperature and contact duration. Moreover, the
surface type may also affect the efficacy of biocides against biofilms [1]. The involvement of surface type is
mostly related to the nanoscale surface morphology which affects the biofilm architecture and weakens the
effectiveness of cleaning and sanitizing procedures [159].

7.1.1.1. The biofilm resistance to disinfectants
Micro-organisms are generally adhered to surfaces under a biofilm state. Disinfectants are often used at very
high concentrations relative to their minimal inhibitory concentrations in order to make it impossible for
bacteria to overcome the massive damage and develop resistance [160]. Many studies have shown that bacteria
exposed to disinfectant levels lower than those required to deliver a lethal insult might develop resistance. In
fact, the cells living under a biofilm state can be up to 1000 fold more resistant to disinfectant agents than their
planktonic counterparts [1]. Thus, the disinfectant agents are frequently inefficient in the eradication of biofilms
and increase the risk of severe health problems and economic losses. In fact, there are many strategies evolved
by biofilm cells to achieve or increase their resistance: (1) Diffusion limitations of disinfectants in biofilms, (2)
The phenotypic adaptations of biofilm cells to sub-lethal concentrations of disinfectants and (3) presence of
disinfectant-adapted and persister cells.
7.1.1.1. 1. Diffusion limitations of disinfectants in biofilms
A mature biofilm is characterized by the production of an extracellular matrix composed of exopolysaccharides
(EPS), proteins and lipids [161,162]. The multiple layers of cells and EPS may constitute a complex and
compact structure which prevents disinfectants from penetrating and reaching the internal layers, thus
hampering their efficacy. It has been shown, that the disinfectant’s diffusion and reaction limitations are
involved in the biofilm resistance [163]. In fact, several studies have found that the restricted diffusion of
disinfectant molecules was related either to the size exclusion or the electrostatic interactions. The interactions
between antimicrobials and biofilm components seem more likely to explain the limitations of penetration into
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